Intercalation-based assay

The discovery of aptamers in 1990 has opened a new scenario in the development of RNA/DNA-based assays for different molecules (Tuerk and Gold, 1990 McGown et al., 1995), but a further advance in nucleic acid nanotechnology was made in 1994 with the first application of a DNA with a catalytic function (DNAzyme) (Breaker and Joyce, 1994). An example of DNAzyme is a single-stranded guanine-rich nucleic acid that has been reported for binding a hemin to yield a DNAzyme possessing peroxidase-like activity. It was proposed that the intercalation of hemin into... 

The low torsion constant at a = —0.025 is very similar to that observed in a supercoiled pBR322 that was partially relaxed by saturation binding of Escherichia coli single-strand binding (ssb) protein, and which persisted for over a month.(56) It is also similar to that recently inferred from an in vivo assay based on variation in repression efficiency with size of a putative DNA loop.(234) Indeed, it appears that anomalously low torsion constants may be universally encountered in the course of either partial or complete relaxation of supercoiled DNAs, regardless of whether the superhelix density is reduced by action of topoisomerase I, binding of ssb protein, binding of intercalated... 

Fluorescence assays are considered among the most convenient, sensitive, and versatile of all laboratory techniques. However, the purine and pyrimidine bases yield only weak fluorescence spectra. Le Pecq and Paoletti (1967) showed that the fluorescence of a dye, ethidium bromide, is enhanced about 25-fold when it interacts with DNA. Ethidium bromide, which is a relatively small planar molecule (Figure El3.4), binds to DNA by insertion between stacked base pairs (intercalation). The process of intercalation is especially significant for aromatic dyes, antibiotics, and other drugs. Some dyes, when intercalated into DNA, show an enhanced fluorescence that can be used to detect DNA molecules after gel electrophoresis measurements (see Chapter 4 and Experiments 14 and 15) and to characterize the physical structure of DNA. Two analyses of DNA will be completed in this experiment ... 

Organic and inorganic intercalators not covalently attached to oligonucleotides were first reported for study of CT in DNA. These experiments provide little information, because of the lack of an accurate measurement of the distance between the D and A and the concern for pairing of the intercalators. A significant experimental improvement came with DNA assays bearing covalently linked intercalators. By use of these systems systematic measurement of distance dependence and base sequence dependence was possible. These experiments led to three important observations ... 

Fluorescence-based indicator reactions can also be coupled to measurements of enzymatic activity. For example, very sensitive assays of endonuclease activity can be performed using as a substrate complexes of double stranded DNA containing intercalated ethidium bromide, which is strongly fluorescent. On cleaving or nicking the DNA, the intercalated ethidium is released into the solution causing a pronounced decrease in fluorescence intensity. 

The DNA lesion 8,5 -cyclo-2 -dG, formed by attack of hydroxyl radicals, contains damage to both base and sugar, and is therefore repaired by nucleotide excision repair enzymes, and is involved in diseases with defective nucleotide excision repair. A mass spectroscopic assay has been developed for the quantitation of the lesion after enzymatic separation of the 5 (R) and 5 (S) isomers. The thermodynamic stability of ODNs containing the oxidative lesion, 2-hydroxy-dA has been examined. It was shown that when the lesion was in the middle of a DNA duplex it behaved as a universal base, in that there was no dilference in Tm when opposite any of the canonical bases. On the other hand, when it was near the termini, there was a preference for base pairing with thymidine, but it also formed base pairs with other nucleotides which was sequence dependent. The extent of oxoprenylation by malondialdehyde or adenine propenal has been investigated in DNA, see (139). ssDNA was found to be more sensitive to oxoprenylation, and supercoiled-DNA more susceptible than linearised plasmid DNA. A variety of intercalators were used, some of which inhibit oxoprenylation, e.g. netropsin, whilst others, like ethidium bromide, caused enhanced oxoprenylation. 

Sensitive fluorometric assays for double-stranded (native) DNA in tissue extracts are based on the noncovalent interaction of intercalating dyes (Fig. 1.7 such as ethi-dium bromide, 4,6 -diamidino-2-phenylindole (DAPI), or 2-(2-[4-hydroxyphenyl]-6-benzimidazolyl)-6- (1-methyl-4-piperazyl)benzimidazol trihydrochloride (Hoechst 33258), with double-stranded DNA in neutral, aqueous solutions. 

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